Highly pathogenic avian influenza (HPAI) virus H5N1 is an emerging infectious pathogen that is capable of causing a worldwide pandemic with widespread debilitating illness and death. There is an urgent need to understand MAPI H5N1 virus evolution and to develop therapeutic strategies to elicit protective host immunity, both passively and actively. In the case of HPAI H5N1, the importance of neutralizing antibodies in preventing disease onset is clearly established. This U01 Project represents the combined efforts of members of the Marasco (Antibody Engineering), Liddington (Structural Biology) and Donis (Molecular Virology) laboratories to investigate and further develop a novel anti-viral strategy that we term "Convergent Combination Immunotherapy (CCI)" for the prevention and early treatment of HPAI H5N1 virus infection. This approach involves the use of a cocktail of neutralizing human Mabs that is specifically formulated to have the broadest neutralization activity against emerging HPAI H5N1 variants. This novel concept is based on the understanding that influenza viruses undergo rapid neutralization escape under immune pressure. Our hypothesis, based on extensive preliminary data, is that it may be possible to focus sufficient immune pressure against critical neutralizing epitopes on hemagglutinin (H5) and neuraminidase (N1) HPAI to prevent neutralization escape or if it occurs, it will be at a great cost to viral fitness and pathogenicity. In this proposal, we will test 14 hypotheses that relate to defining the neutralizing epitopes on these two viral proteins and the effects of immune pressure on influenza virus evolution. In aim 1, we will isolate panels of high affinity human antibodies by phage display against all neutralizing epitopes on HAS and NA1 and will test their cross-neutralization activity using both H5/N1 pseudotype HIV-1 reporter viruses and clade 1/2 H5N1 strains. In aim 2 we will map the neutralization epitopes, determine antibody binding affinities and mechanisms of neutralization and perform co-crystallographic studies. In aim 3, we will perform extensive studies to examine pathways of neutralization escape. Central to our hypothesis, we will also test whether a cocktail of three neutralizing antibodies (CCI) to different neutralizing epitopes or sequential epitopes in a unique escape pathway can prevent neutralization escape. In aim 4 we will examine the cost of neutralization escape on viral fitness in vitro and will test the hypothesis that viruses that escape CCI are less pathogenic in vivo. We anticipate that 12-15 fully neutralizing human Mabs against HPAI H5N1 will be produced from our studies. The final goal of this proposal is to produce a cocktail of three Mabs, termed "TriAIVumab" that can be moved forward in to human clinical trials.